This competitive renewal requests continued support for our investigation on ILK, a central component of supramolecular integrin adhesome involved in regulating diverse cellular processes such as cell shape change, migration, and survival. Discovered in 1996, ILK has long been thought to function as a serine- threonine kinase that phosphorylates cytoplasmic tails (CT) of integrin receptors and transmit signals between ECM and actin cytoskeleton. Highly elevated in failing hearts and tumors, ILK has also been pursued as a hot kinase target for therapeutics development. However, our recent structural analysis revealed that ILK contains a severely degraded active site. Extensive biochemical analyses, in parallel with genetic data, corroborated this observation, strongly indicating that ILK is a pseudokinase. Our finding thus challenges a 16-year dogma, signifying a major new direction of the ILK research as indicated by a dozen recent review articles. Our immediate questions are: (i) without catalytic function, how does ILK mediate the integrin-actin communication and other signaling events? (ii) Given the clinical importance of ILK, what might be a new strategy for the ILK- based therapy? In preliminary studies, we found that ILK utilizes two distinct interfaces, one at its N-terminal ankyrin repeat domain (ARD) and the other at the pseudoactive site of the C-terminal kinase-like domain (KLD), to bind adaptors PINCH and Parvin respectively, leading to the formation of a tight heterotrimer (IPP). We further discovered that this ILK-centered heterotrimer binds G-actin and promotes dynamic F-actin assembly. These findings provide exciting clues for unraveling the mechanism of ILK function. In particular, they potentially explai a long-standing puzzle why loss of ILK drastically impairs the actin cytoskeleton and inhibits cell migration. Since ILK also binds to integrin and other signaling molecules, we are entering into a new era for elucidating how ILK may act as a central scaffolding machinery to regulate cytoskeleton remodeling and cell adhesion. In the next funding period, we will rigorously examine this machinery by determining the detailed structural basis of ILK-mediated actin assembly via IPP (Aim1) and elucidate how this actin assembly process is coupled with ECM via a novel integrin-IPP-actin pathway (Aim2). We will further investigate how this pathway may cross-talk with other pathways for cooperative regulation of cell adhesion dynamics (Aim3). Overall, our proposal represents a major programmatic shift for the ILK research. The results will provide a new and comprehensive view of the ILK-mediated signaling and cell adhesion. They may also impact on our understanding of multiple ILK-associated diseases and also re-invigorate strategies for the ILK-targeted therapy.